The phenotypic expression of a transgene in a plant is determined both by the structure of the gene itself and by its location in the plant genome. At the same time the presence of the transgene at different locations in the genome will influence the overall phenotype of the plant in different ways. The agronomically or industrially successful introduction of a commercially interesting trait in a plant by genetic manipulation can be a lengthy procedure dependent on different factors. The transformation and regeneration of genetically transformed plants are only the first in a series of selection steps that include extensive genetic characterization, breeding, and evaluation in field trials.
Oilseed rape (OSR) (Brassica napus, AACC, 2n=38) is a natural hybrid resulting from the interspecies hybridization between Cole (Brassica oleracea, CC, 2n=18) and Turnip (Brassica campestris, AA, 2n=20). Winter oilseed rape is sown during the last 10 days of August and the first ten days of September and harvested the following July, needing a temperate period for vernalization. The faster growing spring rapes are sown during late March and early April being harvested mid August to September. The main types of OSR grown at present are low and high erucic acid varieties. Double low (00) varieties contain low (typically less than 1%) levels of erucic acids (which humans find hard to digest), and low levels of glucosinolates (which makes the meal by-product indigestible for animals). Current uses for “00” varieties include oil for human consumption and high protein meal for animal feed. Industrial uses include feedstocks for pharmaceuticals and hydraulic oils. High erucic acid rape (HEAR) varieties are grown specifically for their erucic acid content—typically 50-60% of oil. The principal end use of HEAR is to produce erucamide, a “slip agent” used in polyethane manufacture. A small portion is used to produce behenyl alcohol, which is added to a waxy crude mineral oil to improve its flow.
Oilseed rape plants are bisexual and typically 60-70% self-pollinated. The production of hybrids and introduction of genetic variation as a basis for selection was traditionally dependent on the adaptation of natural occurring phenomena such as self-incompatibility and cytoplasmic male sterility. Artificial pollination control methods such as manual emasculation or the use of gametocides are not widely applied in OSR breeding due to their limited practicability and high cost respectively.
Transgenic methods have been developed for the production of male or female-sterile plants, which provide interesting alternatives to the traditional techniques.
EP 0,344,029 describes a system for obtaining nuclear male sterility whereby plants are transformed with a male-sterility gene, which comprises, for example a DNA encoding a barnase under the control of a tapetum specific promoter, PTA29, which when incorporated into a plant ensures selective destruction of tapetum cells. Transformation of tobacco and oilseed rape plants with such a chimeric gene resulted in plants in which pollen formation was completely prevented. Mariani et al. (1990) Nature 347:737-741.
To restore fertility in the progeny of a male-sterile plant, a system was developed whereby the male-sterile plant is crossed with a transgenic plant carrying a fertility-restorer gene, which when expressed is capable of inhibiting or preventing the activity of the male-sterility gene (U.S. Pat. No. 5,689,041, U.S. Pat. No. 5,792,929). Such a fertility-restorer gene is placed under the control of a promoter directing expression at least in the cells in which the male-sterility gene is expressed. Mariani et al. ((1992) Nature 357:384-387) demonstrated that the sterility encoded by the pTA29:barnase gene can be restored by the chimeric pTA29:barstar gene in oilseed rape.
Cytochemical and histochemical analysis of anther development of B. napus plants comprising the chimeric pTA29:barnase gene alone or with pTA29:barstar is described by De Block and De Brouwer ((1993) Planta 189:218-225).
Successful transformation of Brassica species has been obtained by a number of methods including Agrobacterium infection (as described for example in EP 0,116,718 and EP 0,270,882), microprojectile bombardment (as described for example by Chen et al. (1994) Theor. Appl. Genet. 88:187-192) and direct DNA uptake (as described for example by De Block et al. (1989) Plant Physiol. 914:694-701; and Poulsen (1996) Plant Breeding 115:209-225.
However, the foregoing documents fail to teach or suggest the present invention.